Two-photon Induced Fluorescence Research Articles

Two-Photon-Induced Fluorescence Study of Rhodamine-6G Dye in Different Sets of Binary Solvents.

This study deals with the effects of different sets of binary solvents on the Two-Photon Induced Fluorescence (TPIF), a non-linear process, of the Rhodamine-6G (Rh6G) dye, which is a well-known xanthene dye. This work examines the importance of inter-molecular interactions, which results in the modulation of the TPIF of the Rh6G. In this work, we have investigated three binary solvent mixtures representing varying polarity and intermolecular interactions. Specific solvent mixtures used are methanol-water, methanol-dimethyl formamide, and methanol-chloroform. Since the solvent polarity across these binary solvents differs, there are significant intermolecular interactions in the binary mixture solvents, which modulate the two-photon process of Rh6G when irradiated with high-intensity laser light at 780nm. In our studies, we find that Rh6G in the MeOH-H2O binary solvent has maximum red-shift and minimum intensity as compared to other pairs of binary liquids when the volume fraction of methanol decreases due to more extensive hydrogen bonding between the two components. Additionally, at 1:1 ratio of binary mixtures, Rh6G is found to have the highest TPEACS value for methanol-chloroform binary solvent and reason for that is related to the formation of weak H-bond networks between proton donor chloroform and proton acceptor methanol.

Concentration Dependent Approach for Accurate Determination of Two-Photon Absorption Cross-Section of Fluorescent dye Molecule.

We have investigated the concentration dependent of two-photon induced fluorescence (TPIF) in methanolic solution of Rhodamine 6G and Rhodamine B dye using 120fs laser pulses at 780nm, 76MHz repetition rate. TPIF study of these dyes was compared with their respective one photon fluorescence intensity. We have shown the effect of chopper on TPIF intensity from Rhodamine dyes, which have shown direct influence on the determined TPA Cross section of these dyes.

PH Effect on Two-Photon Cross Section of Highly Fluorescent Dyes Using Femtosecond Two-Photon Induced Fluorescence.

Effect of solution pH on two-photon absorption cross-section of highly fluorescent Coumarin and Rhodamine dyes with high repetition rate femtosecond laser pulses at 780nm is presented using two-photon induced fluorescence technique. A correspondence in the measured two-photon and single-photon cross-section values is seen when the pH changes from acidic to basic conditions (pH=2-10) for solutions in 1:1 water-ethanol binary mixture. By plotting changes in the single-photon and two-photon fluorescence in this pH range, the excited state pKa values are found. The ground state pKa values are also affected by the protonation deprotonation equilibrium as a result of variation in pH from acidic to basic, which are characterized by changes in absorbance spectra. Most of these single-photon and two-photon induced fluorescence spectra show characteristic blue shifts. Different fluorescence quantum yields calculated at each pH reflect a change in structure corresponding to their associated properties as a result of acid base equilibrium.

Precise autofocus method employing normalized fluorescence image size in a two-photon polymerization nanofabrication system.

The autofocus method has been investigated to improve the precise positioning of a substrate surface at the center of the laser focal spot in two-photon polymerization (TPP) nanofabrication. For this purpose, we developed a novel autofocus method using normalized image size, which was calculated with the second momentum radius (SMR) of two-photon induced fluorescence (TPIF). The SMR of TPIF was theoretically analyzed and experimentally compared with the average intensity of TPIF for various input laser powers. The results show that the proposed method enhanced the precision and robustness of autofocus in TPP. Specifically, the experimental creation of ascending voxel arrays demonstrated both the method's immunity to input laser power change, and a high precision of ±0.045 μm. To test the practical feasibility of the proposed autofocus method, 300 μm×260 μm single-layer honey-comb structures were successfully fabricated with precompensation and dynamic compensation using the proposed autofocus method.

INFLUENCES OF AGGREGATION ON THE TWO-PHOTON FLUORESCENCE PROCESS

Rhodamine B (RhB) is most often used as the reference in the two-photon induced fluorescence (TPIF) method to measure the two-photon absorption (TPA) cross-section. In this work, we investigate the influences of dimer formation, energy transfer and self-absorption on the two-photon fluorescence process. Those three effects will result in a large measurement error of the TPA cross-section. We thus provide a concentration gradient correction method by considering the above three processes to eliminate the measurement error.

Sensing of Mycotoxin Producing Fungi in the Processing of Grains

Due to the growing demand for rapid, sensitive and inexpensive detection of contaminations in food and feed, both in the field and after harvest, non-invasive and non-destructive spectroscopic methods are reviewed for the direct identification of fungi and mycotoxins on grain and in grain products. Different model samples containing defined contaminations of fungi and/or mycotoxins as well as real-world samples were investigated using spectroscopic methods (steady-state and time-resolved fluorescence spectroscopy, ultraviolet/visible/near infrared (NIR) spectroscopy, two-photon-induced fluorescence (TPIF)). To evaluate the capability of TPIF for on-line and in-line mycotoxin analysis in real-world samples, fundamental parameters of different mycotoxins, such as two-photon cross-sections, were determined in beer and wine. A comparison showed that the disturbance due to background emission originating from matrix constituents is significantly reduced under two-photon excitation conditions, and TPIF can be applied for a qualitative and quantitative analysis of mycotoxins in wine and beer. The combination of data obtained from different spectroscopic methods (such as optimised excitation and emission wavelengths, fluorescence decay times and fluorescence quantum efficiency on the one hand side and NIR spectroscopy on the other side) is promising for the qualitative as well as quantitative identification of fungi and mycotoxins. Moreover, NIR reflection spectra yield additional information on ingredients, moisture content and the presence (or absence) of fungi in the sample. The spectroscopic techniques are complemented by chemometric tools to extract the desired chemical information, e.g. with respect to the presence of contaminations.

Two-Photon Absorption Property of a Conjugated Polymer: Influence of Solvent and Concentration on Its Property

The Suzuki coupling reaction was utilized to prepare target conjugated polymer P1, whose main chain consists of 3,5-dicyanopyridine moieties as electron acceptors and both triphenylamino groups and fluorene moieties as donors. This polymer exhibits excellent solubility in organic solvents and high thermal stability (5% of weight loss at 398 degrees C). Detailed spectroscopic studies including absorption and fluorescence emission in solvents of different polarity were also conducted. The influence of the solvent and concentration on its two-photon absorption (TPA) property was studied by a two-photon-induced fluorescence (TPIF) method. It was found that the highest two-photon absorption cross section (13,260 GM at a laser wavelength of 940 nm) appeared in 1,2-dichloroethane, a solvent with a moderate polarity. Meanwhile, with the increase of its concentration in chloroform, its TPA cross section value is reduced, implying that its two-photon absorption process in its chloroform solution is not a pure intrachain process.

Water-Soluble Two-Photon Fluorophores Based on Paracyclophane

We report the synthesis, linear and nonlinear spectroscopy of the paracyclophane-based two-photon absorption (TPA) fluorophores. Two-photon induced fluorescence (TPIF) measurements in water show a substantial decrease in TPA cross section (δ) and fluorescence quantum yield (η), relative to toluene. We note that to obtain optimum two-photon action cross sections (ηδ) in water, one needs to fine tune the intramolecular charge transfer (ICT). Microenvironment modulation using the micellar structure in water increases ηδ values similar to those obtained in toluene. The measured ηδ values in water are the highest among reported so far, suggesting important guidelines for designing of optimized TPIF molecular-labels.

Polarity Probing Two-Photon Fluorophores Based on [2.2]Paracyclophane

A series of tetra donor substituted [2.2]paracyclophane-based two-photon absorption (TPA) fluorophores were synthesized in neutral and cationic forms. The imaging activity of overall set of fluorophores was studied by the two-photon induced fluorescence (TPIF) method in a range of solvents. We also measured a clear progression toward a longer photoluminescence lifetime with increasing solvent polarity (intrinsic photoluminescence lifetime, τi: ~2 ns in toluene → 12-16 ns in water). The paracyclophane fluorophores with this unique property can be utilized as an optical polarity probe for the biomolecular substrates. The combined measurement of the two-photon fluorescence microscopy (TPM) cell image and TPIF lifetime can give us a better understanding of the biological processes and local environments in the cells.

Two-photon absorption cross-sections and time-resolved fluorescence imaging using porphyrin photosensitisers

Three porphyrin systems have been characterised for use in two-photon fluorescence imaging of biological samples. We have determined the two-photon absorption cross sections (sigma(2)) of the di-cation, free-base and metallated forms of hematoporphyrin derivative (HpD), hematoporphyrin IX (Hp9) and a boronated protoporphyrin (BOPP) using the open-aperture Z-scan and the two-photon induced fluorescence (TPIF) techniques at an excitation wavelength of 800 nm. The insertion of either protons or a metal ion into the macrocycle is shown not to significantly influence the sigma(2) of the porphyrins. Two-photon time-resolved fluorescence images of C6 glioma cells transfected with a free-base form of the BOPP have been obtained as a function of the porphyrin concentration. These studies reveal a maximum useful porphyrin concentration for fluorescence imaging purposes of approximately 30 microg mL(-1).

Two-photon absorption and up-converted fluorescence of novel triphenylamine-based oligomers

Three new conjugated oligomers (referred to as P1, P2 and P3), of “D–π-A” structure with the same donor group of 4-methyl triphenylamine but different acceptor groups such as naphthalene for P1, dibenzothiophene for P2, and dibenzofuran for P3, were synthesized and characterized. The linear optical properties, including absorption and one-photon induced fluorescence, fluorescence quantum yield ( Φ) and lifetimes of the oligomers are reported. Two-photon absorption (TPA) properties were measured by open-aperture Z-scan techniques, while two-photon induced fluorescence (TPF) behaviour was recorded using a CCD spectrosensitometer, employing a Ti:sapphire femtosecond laser pulse. It was found that P1 displayed the greatest two-photon absorption coefficient ( β) due to its larger intermolecular charge transfer and stronger interactions between donor and acceptor groups. In contrast, P3 exhibited strongest one- and two-photon fluorescence due to its dibenzofuran acceptor group possessing more preponderant radiation dynamics, in comparison with its dibenzothiophene- and naphthalene-based counterparts.

Synthesis and structure–photophysical property relationships for two coumarinyl-based two-photon induced fluorescent molecules

The synthesis and the structure–photophysical property relationships for two new coumarinyl-based organic heterocyclic molecules are presented. These molecules consist of a typical A–π–D or A–π–A′ structure, where 3-carbonylcoumarinyl, vinyl and anthryl (or indolyl) groups are employed as acceptor (A), π-conjugated center (π) and donor (D) (or the other acceptor, A′) moieties, respectively. Based on steady-state as well as time-resolved techniques, it is found that this new kind of π-conjugated framework consisting of a 3-carbonylcoumarinyl acceptor and a vinyl π-conjugated center is a useful conjugated π-electron building block with potential applications in forming new kind of effective organic two-photon induced fluorescence (TPIF) molecules. Additionally, experimental results confirm that remarkable enhancements in the fluorescence quantum yield and TPIF activity can be obtained by replacing anthryl group with an indolyl group in the terminal moiety of the coumarinyl-based π-conjugated framework.

Fluorescence Studies of Melanin by Stepwise Two-Photon Femtosecond Laser Excitation

Fluorescence of synthetic melanin in the solvents H2O, KOH, ethylene glycol monomethyl ether, and dimethyl sulfoxide has been excited by two-photon absorption at 800 nm, using 120-fs pulses with photon flux densities of ≥1027 cm−2.S−1. Compared to the one-photon (400-nm)-induced fluorescence of melanin, the overall spectral shape is red-shifted and shows a strong environment sensitivity. The decay of the two-photon-induced fluorescence (TPF) of melanin is three-exponential, with a shortest main component of about 200 ps. The results of the TPF studies in line with the unique light absorption property of melanin of a monotonously decreasing absorption spectrum between the near UV-region and the near infrared region indicate that the TPF is realized via stepwise absorption of two 800-nm photons. In comparison to the simultaneous absorption of two photons, the stepwise process needs lower photon flux densities to get a sufficient population of the fluorescent level. This stepwise process offers new possibilities of selective excitation of melanin in skin tissue in a spectral region where there is no overlap with any absorption of another fluorescent tissue component. The first results with different samples of excised human skin tissue (healthy, nevus cell nevi, malignant melanoma) suggest that fluorescence excited in this way yields information on malignant transformation.

Effects of Two-Photon Fluorescence and Polymerization on the First Hyperpolarizability of an Azobenzene Dye

The technique of hyper-Rayleigh scattering (HRS) has been employed to investigate the first hyperpolarizability (β) of the azo dye Disperse Red 19 and a polymer in which Disperse Red 19 appears as a side-chain moiety. In addition to HRS, these two molecules were found to exhibit substantial two-photon-induced fluorescence (TPF). The TPF spectra for these molecules were measured, and corrected HRS intensities were obtained. Using an external reference method, β values of (1480 ± 100) × 10-30 and (1470 ± 90) × 10-30 esu were determined for the dye and polymer systems, respectively, in dimethyl sulfoxide at 1064 nm. The difference between these two values is very small and shows that the effect of polymerization on the HRS intensity, in the dilute concentration limit, is negligible.